Abstract: Aeromonas hydrophila, gram negative facultative anaerobic short bacillus, causes red fin disease, haemorrhagic septicemia, motile aeromonad septicemia and other infections in Carassius auratus. Multiple drug resistance of some strains of A. hydrophila presents a major threat in the control these diseases. Recently much attention has been paid to extracts and biologically active compounds isolated from plant species. In this studies ten plants leaves were assayed against Aeromonas hydrophila both in vitro and in vivo. The effective plant was found to be Phyllanthus emblica which shows the 24 mm zone diameter which was the nearest to oxytetracyclin and further choose for the in vivo study. Tagetes erect, Azadirachta indica, Calotropis procera also showed nearer activity nearer to the control antibiotic. There were no effect of Momordica charantia, Achyranthes aspera, Centella asiatica on aeromonads disease as the solvent alcohol solely gave the zone diameter of 14 mm. When incorporated P. emblica leaf extract in infected gold fish at a dose of 25 mg g-1 of body weight, the A. hydrophila infection was cured within 12-15 days under laboratory condition.
INTRODUCTION
Aeromonas hydrophila causes red fin disease, haemorrhagic septicemia, motile aeromonad septicemia and other infections by in gold fish, Carassius auratus. The organism Aeromonas hydrophila is a gram negative facultative anaerobic short bacillus. It is motile and grows wide range of temperature 11.2-40.5°C. It produces exotoxin which exhibit toxicity to the carp. A. hydrophila infection is a zoonotic disease. It causes bacteremia, cellulites, myonecrosis, ecthyma gangrenosum in human. The Gold Fish, Carassius auratus is an ornamental fish contain the golden color pigment due to the presence of erythrophore pigments. This fish has high susceptibility to motile aeromonads and are commonly valuable for experimental animals.
For this studies 10 plant species were selected, which was based on their use by traditional Indian healers or their reported antimicrobial activities in an attempt to find bioactive medicines for use in the treatment haemorrhagic septicemia, skin ulsers and wounds. The plants are Phyllanthus emblica, Tagetes erecta, Azadirachta indica, Calotropis procera, Aloe vera, Citrus limon, Carum copticum, Momordica charantia, Achyranthes aspera, Centella asiatica. The plant part chosen was leaves. The leaf of this plant contains tennins, Polyphenols, corilagin, alkaloids, phyllantidine, flavonoids etc as antimicrobials.
To control bacterial diseases in fishes usually the chemotherapeutic agents like chloramphenicol, tetracycline, streptomycin etc are used. There are wide ranges of current antibiotics that are used for the treatment of bacterial infections but are still some challenges to be met in microbial chemotherapy. One of the problems in the development of resistance of chemotherapeutic agent is due to abuse of these drugs. Some strains of A. hydrophila show multiple drug resistances. Some time the chemotherapeutic agents also accumulate inside various organs of fish and become toxic to them. Because of the side effects and the resistance that pathogens build against antibiotics, recently much attention has been paid to extracts and biologically active compounds isolated from plant species. The use of plant extracts, as well as other alternative forms of medicinal treatments, is enjoying great popularity in late 1990s (Cowan, 1999). The acetone extracts of the roots of Rumex crispus and Acinos rotundifolius demonstrated significant inhibitory effects against A. hydrophila (Ulukanli et al., 2005). Studied the efficacy of plant extracts in inhibiting Aeromonas hydrophila and Listeria monocytogenes in refrigerated cooked poultry (Hao et al., 1998).
The main objective of this research was to determine the phytochemicals effectiveness of the plants extracts against the microorganism which cause diseases in the gold fish by both in vitro and in vivo. The antibiotic sensitivity test was determined only by agar well diffusion method and it gave clear zone of inhibition. These results were compared with the potent antibiotics that are effective against these diseases. In vivo studies of the infected fish were carried out with the most effective plant extract with the feed.
MATERIALS AND METHODS
Collection of Samples
Infected live fishes were anaesthetized by using chloroform. Innoculum from
the infected part of the fish was taken out under aseptic conditions and the
infected part was inoculated into nutrient broth and incubated at 27°C for
24 h.
Isolation, Screening, Selection and Identification of Aeromonas hydrophila
The isolation of Aeromonas hydrophila was carried out with Rimler
Shotts agar plate. One milliter of the preinoculam was plated and incubated
at 27°C for 24 h in an inverted position. The colonies were screened and
biochemically identified as Aeromonas hydrophila (Bergey’s Manual)
(Holt et al., 2000).
Plant Material and Preparation of Extracts
The plant material used in this study consisted of Phyllanthus emblica,
Tagetes erecta, Azadirachta indica, Calotropis procera,
Aloe vera. Citrus limon, Carum copticum, Momordica charantia,
Achyranthes aspera and Centella asiatica which were collected
from different parts of Bangalore and Dharwad, Karnataka, India during October
2005. For 8 plant species fresh leaves were washed under running tap water,
air dried and shade dried for three weeks. Calotropis procera and Carum
copticum leaves were thick so sun dried carried out. Then the leaves were
grounded using blender to get the powdery form.
Solvent Extraction
Twenty five gram of dried plant was extracted with 100 mL of 70% alcohol
in conical flasks sealed with foil and allowed to stand for 72 h. They were
filtered with Whattman Filter paper No. 1 to obtained crude ethanolic extracts
and stored at 4°C when not in use.
Antibacterial Assay
The antimicrobial properties were evaluated by the agar well diffusion method
using Mueller Hinton agar (Hi-media) for the assay. The microorganisms were
activated by inoculating a loopful of the strain in the nutrient broth (25 mL)
and incubated at 27°C for 24 h.
One milliter of inoculum was inoculated into the 45-50°C cooled agar and plated. Using the cork borer wells was made and different alcohol extracts having 25 mg mL-1 concentration were transferred using a micropipette. Then the plates were kept in the refrigerator for 5 min for diffusion and incubated at 27°C for 24 h.
The control experiment was carried out with oxytetracyclin of Ranbaxy Company, India i.e., the drug of choice for the aeromonad infection and solvent alcohol.
Animal Testing
Ten gold fish (GF1-GF10) were obtained local fish aquarium centers and maintained
in the lab condition. Isolated and identified Aeromonas hydrophila were
cultured on Rimler Shotts agar medium and further diluted from 10-1
to 10-15 dilutions in saline water were carried out. Ten fishes were
injected at the caudal peduncle region with 0.5 mL the serially diluted solution
10-6 to 10-15, respectively with 1 mL disposable insulin
syringe. The fishes after being inoculated with the inoculum showed several
symptoms like lack of appetite, swimming abnormalities, pale gills, blotted
appearance and skin alliterations. The leaf extract was mixed with the fish
feed at dosages of 25 mg g-1 of body weight of fish and feed daily
for a period of 12-15 days.
RESULTS AND DISCUSSION
The organism isolated from the fish was confirmed as Aeromonas hydrophila shown (Table 1).
The antibacterial activities of the extracts obtained from the plants under study and the standard antibiotic was determined by the agar well diffusion method (well diameter 6 mm) was shown in the Table 2.
The effective plant was found to be Phyllanthus emblica which shows the 24 mm zone diameter which was the nearest to oxytetracyclin and further choose for the animal study. T. erect, A. indica, C. procera also showed nearer activity nearer to the control antibiotic. There were no effect of M. charanti, A. aspera, C. asiatica on aeromonads disease as the solvent alcohol solely gave the zone diameter of 14 mm.
The in vivo studies showed the days of recovery of the fish from the disease and shown in Table 3.
P. emblica leaf extract has the healing capacity for the A. hydrophila infection of the gold fish at a dose of 25 mg g-1 of body weight of the fish for a period of 12-15 days under laboratory condition.
| Table 1: | The biochemical characteristics of Aeromonas hydrophila |
| Table 2: | Inhibitory properties (inhibition zone diameter in mm) of different plant extract on Aeromonas hydrophila |
| Table 3: | Effect of oxytetracyclin and P. emblica leaf extract (25 mg g-1 b.wt.) in vivo |
Multiple drug resistance of some strains of A. hydrophila presents a major threat in the control of the motile aeromonad septicemia in fish and much attention has been paid to extracts and biologically active compounds isolated from plant species. In this study the plant species Phyllanthus emblica leaf extract showed the same antimicrobial activity as compare to the drug of the choice, oxytetracyclin. The days of recovery from the disease also only 1.2-1.5 times less compared to the control. The Phyllanthus emblica leaves contain tennins, polyphenols, terchebin, terchebin corilagin, alkaloids like phyllantidin and phyllantine which acts as antimicrobial to kill or inhibit the bacteria.
Result of this kind herald an interesting promise of designing a potentially active anti bacterial agent of plant origin. Many plants have become sources of important drugs and the pharmaceutical industries have come to consider traditional medicine as a source of bio-active agents that can be used in the preparation of medicine (Aboaba et al., 2006).
The present study confirmed the antibacterial effect of the Phyllanthus emblica leaf extract which can be treated apart from the synthetic drug to overcome the toxicity and the resistance of the bacteria. But this study was confined only for laboratory conditions hence it can also be further considered for studying the inhibitory action in natural ecosystem for the commercial usages.
ACKNOWLEDGMENT
We are very much grateful to Dr. Raju K.C., Head of the Department, Department of Microbiology, Administrative Management College, Bangalore for his valuable guidance and support.